Urinary excretion of biomarkers of oxidative kidney damage induced by ferric nitrilotriacetate.

There is an increasing need for biomarkers of oxidative stress in animals and man. In this study, we have evaluated in the rat the utility of various endogenous products that are excreted in urine as potential noninvasive biomarkers of oxidative stress in the kidney. Renal oxidative damage was induced by daily i.p. injections of ferric nitrilotriacetate (Fe-NTA) for a period of 13 days. The daily dose of Fe-NTA was increased during the experiment from 6 to 40 mg Fe/kg body wt. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), coproporphyrin III (COPRO III), seven aldehydes, and acetone were determined in fractionated urine samples and compared with commonly used urinary and plasma clinical chemical parameters for toxicity. The parameters that showed the earliest increase were acetaldehyde (ACET), propanal (PROPA), and COPRO III. Their increase was significantly earlier than that of classical clinical chemical parameters indicative of renal damage such as urinary concentration of glucose (GLU) and protein (PRT), and N-acetyl-beta-D-glucosaminidase (NAG) activity. The excretion of 8-OHdG was increased only after administration of the highest dose of Fe-NTA. Urinary excretion of acetone, form-aldehyde (FOR), butanal (BUTA), pentanal (PENTA) hexanal (HEXA), and malondialdehyde (MDA) was also increased; however, their increase occurred only slightly before or simultaneously with that of the urinary clinical chemical parameters. In conclusion, 8-OHdG, acetone, FOR, BUTA, PENTA, HEXA, and MDA may possibly serve as biomarkers for oxidative kidney damage. COPRO III, ACET, and PROPA might even be used as biomarkers of production of reactive oxygen species at an early stage.

Urinary excretion of biomarkers for radical-induced damage in rats treated with NDMA or diquat and the effects of calcium carbimide co-administration.

The urinary excretion of seven aldehydes, acetone, coproporphyrin III and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) as non-invasive biomarkers of oxidative damage was measured in rats treated with diquat or N-nitrosodimethylamine (NDMA), two compounds causing hepatic damage by different mechanisms. Furthermore, the effect of co-administration of the aldehyde dehydrogenase inhibitor, calcium carbimide (CC) on the urinary excretion of the aldehydes was determined. Slight hepatotoxicity was found at the end of the experiment after treatment with NDMA (0.5, 4 and 8 mg/kg at t = 0, 48 and 96 h, respectively) or diquat (6.8 and 13.6 mg/kg at t = 0 and 48 h, respectively). In diquat treated rats slight nephrotoxicity was also found. Urinary excretion of aldehydes, acetone and coproporphyrin III remained largely unchanged in rats treated with NDMA. In the rats treated with diquat, the urinary excretion of several aldehydes was several-fold increased. An increase was also found in the urinary excretion of 8-OH-dG after the second dose of diquat. Treatment of rats with CC did not significantly influence the urinary excretion of aldehydes in control and NDMA rats. However, in rats treated with diquat, CC caused a potentiating effect on the excretion of acetaldehyde, hexanal and malondialdehyde (MDA), indicating that oxidation of aldehydes to carbonylic acids by aldehyde dehydrogenases (ALDHs) might be an important route of metabolism of aldehydes. In conclusion, increased urinary excretion of various aldehydes, acetone, coproporphyrin III and 8-OH-dG was observed after administration of diquat, probably reflecting oxidative damage induced by this compound. No such increases were found after NDMA administration, which is consistent with a different toxicity mechanism for NDMA. Therefore, excretion of aldehydes, acetone, coproporphyrin III and 8-OH-dG might be used as easily accessible urinary biomarkers of free radical damage.

Application of scanning electron microscopy and energy-dispersive X-ray analysis to the solution behaviour of Zn-insulin: precipitation phenomena.

Scanning electron microscopy (SEM) has been applied in combination with energy dispersive X-ray analysis (EDAX) to identify and analyse particles or particulate matter, occasionally present in clear neutral Zn-insulin solutions. SEM photographs revealed the existence of three different types of precipitate, consisting of particles with a crystalline, amorphous or gel-like nature, respectively. At present, it is not clear which conditions lead specifically to each of these three types of precipitate. The advantages of the EDAX method are shown. The technique enables semi-quantitative analysis to be performed on a single particle as small as 0.2 microns. It was demonstrated with the EDAX method that the particles occasionally found in clear Zn-insulin solutions contain insulin as well as Zn in roughly the same ratio as in the insulin starting material. It is concluded that the EDAX method has great potential in pharmaceutical technology, inter alia for the analysis of emulsion systems (in the frozen state), as well as suspensions and particulate matter in injection fluids. This technique is particularly useful in the latter case, due to its applicability to extremely small sample sizes.

Demonstration of a tertiary interaction in solution between the extra arm and the D-stem in two different transfer RNA's by NMR.

According to the X-ray structure of yeast tRNAPhe at 2.5 A resolution, a hydrogen bond is formed between m7G46 and G22. By removal of this m7G46-residue we demonstrate that this interaction is present in solution as well. Comparison of the 1H 360 MHz NMR spectra of intact yeast tRNAPhe and its m7G-excised derivative locates the position of this tertiary H-bond at 12.5 ppm downfield from DSS. Additional evidence for the presence of this interaction in solution comes from a comparison of 1H NMR spectra of E. coli tRNAf1Met and E. coli tRNAf3Met, which differ only in a single position in the extra arm. In tRNAf3Met residue 47 is a m7G-residue, whereas in tRNAf3Met it is A, resulting in the absence of the m7G47 - G23 - C13 triple interaction, characteristic of tRNAf1Met. The resonance position of this tertiary interaction in tRNAf1Met is located around -13.6 ppm, a chemical shift difference of 1.1 ppm with respect to the position observed for tRNAPhe. The origin of this chemical shift difference is discussed in relation to the structure of their respective augmented D-helices.

Studies of yeast phenylalanine-accepting transfer ribonucleic acid backbone structure in solution by phosphorus-31 nuclear magnetic resonance spectroscopy.

Approximately 17 diester phosphates from the backbone structure of yeast tRNAPhe give rise to phosphorus resonances, which are resolved in its 31P NMR spectrum. To localize these diester phosphates within the tRNA structure, 31P NMR spectra of several chemically or enzymatically modified yeast tRNAPhe species were recorded. To this end selective modifications were performed in the anticodon, the DHU, and the T psi C loop. Modifications, performed in different loop regions, give rise to perturbation of different characteristic 31P resonances. The 31P spectra were correlated with the corresponding 1H NMR spectra of the ring N hydrogen-bonded protons and interpreted in view of the X-ray results obtained on yeast tRNAPhe. It is concluded that the diester phosphate groups, which experience an unusual shift, can be accounted for in the X-ray structure in terms of hydrogen-bonded phosphates groups and diester phosphates with a diester geometry, deviating from the normal double-helical conformation.

The cryopreservation of liposomes. 1. A differential scanning calorimetry study of the thermal behavior of a liposome dispersion containing mannitol during freezing/thawing.

The thermal behavior of water in liposome dispersions and in liposome dispersions containing mannitol at subzero temperatures was investigated with differential scanning calorimetry (DSC). The cooling curves from 20 down to -60 degrees C for a liposome dispersion (bilayer composition PL100H/DCP), monitored at cooling rates of 5 and 10 degrees C/min, showed several heat flows related to water crystallization. All lipid-containing dispersions showed water crystallization at temperatures below -40 degrees C. The magnitude of this heat flow strongly depended on the experimental variables. Cooling rate, particle size, lipid concentration, and location and nature of the cryoprotectant all influenced the water crystallization behavior as shown in the DSC cooling curve. Different fractions of water--presumably related to their location in the dispersion--could be distinguished. It is concluded that DSC provides a valuable tool for the detection of changes in the physical state of water in liposome dispersions during freezing/thawing. The insights gained from these DSC studies may make it possible to select--on the basis of rational considerations rather than by trial and error--optimum conditions for the cryopreservation of liposomes containing water-soluble drugs.

Hydrogen-1 and phosphorus-31 nuclear magnetic resonance study of the solution structure of Bacillus licheniformis 5S ribonucleic acid.

The conformation of Bacillus licheniformis 5S RNA in solution has been studied by using 360-MHz 1H NMR and 40.5-MHz 31P NMR spectroscopy. The 1H NMR spectra, which are well resolved, have been compared with theoretical spectra derived by ring-current shift calculations for various models proposed in the literature for the secondary structure of 5S RNA. The total amount of base pairs is estimated to be around 36. NMR melting experiments indicate that both the molecular stalk and the prokaryotic loop [Fox, G. E., & Woese, C. R. (1975) Nature (London) 256, 505] are present in the solution structure. On this basis, some models proposed for the secondary structure of 5S RNA not containing these structural features can be rejected. Several resonances are observed around 10.7 ppm that can be ascribed to protons involved in non-Watson-Crick base pairing most likely present in tertiary interactions in the 5S RNA molecule or to ring N protons of nonpaired bases which as a result of the molecular folding are shielded from the solvent. Under our solution conditions, these structural features disappear at physiological temperature, the process being uncoupled from the collapse of the secondary structure. Using 31P NMR, we demonstrate that the number of phosphate conformations in the sugar phosphate backbone of 5S RNA, deviating from the g-,g- conformation normally found in double helices, is far les than in tRNA.

Conformational changes of yeast tRNAphe as monitored by 31P NMR.

The 31P NMR spectra of tRNAs contain approximately 17 resonances resolved from the main resonance which consists of about 80% of the total resonance intensity arising from the sugar phosphate backbone. In the present paper we study the behavior of the 31P resonances of yeast tRNAPhe as a function of temperature and of solution conditions. By comparison with other melting experiments we show that three resonances (called c, e and j2) belong to phosphates in the anticodon loop, while the remaining resolved 31P resonances come from phosphates in specific conformations in the central part of the molecule imposed by the tertiary structure. These conformations are different from the normal g-,g- conformation found in A-RNA double helices. The assignments are in good agreement with those previously made on the basis of chemical and enzymatic modification experiments [P. J. M. Salemink, T. Swarthof & C. W. Hilbers (1979) Biochemistry, 18, 3477-3485]. AT high Mg2+ concentrations the anticodon loop is found to be present in two different conformations. For all solution conditions studied loss of the anticodon loop structure takes place before the tertiary structure is melted out. The melting of the tertiary structure is not strictly an all- or-none process. The lifetimes of phosphate conformations involved in the tertiary structure may differ by at least a factor of two. It can also be concluded that the range of chemical shifts observed for phosphodiesters cannot at the moment be accounted for by theoretical calculations.

The influence of gamma-irradiation upon the chemical and biological properties of insulin.

Partially purified insulin preparations of bovine and porcine origin, were subjected to gamma-irradiation with doses ranging from 1.0 up to 25 kGy (0.1-2.5 Mrad) at 0 degrees C or ambient temperature. The susceptibility of insulin to the irradiation was determined by chromatography, electrophoresis and assay of the biological activity. The sterilizing effect of the gamma-irradiation was investigated for Bacillus pumilus as well as for artificial mixtures of lactose and several bacilli. It is concluded that the sterilizing dose for the investigated insulins was greater than or equal to 2.2 kGy. At doses up to 25 kGy at 0 degree C no specific radiolytic products were detectable, whereas the biological activity was fully retained. The content of dimers and the content of related peptides appeared to increase gradually with the irradiation dose absorbed. No effects of long-term storage could be demonstrated on biological and chemical properties of insulin after 2.2, 4.5 and 7.5 kGy.

Pharmacokinetics and miscibility of 4 highly purified porcine insulins: a study in vitro and in healthy volunteers.

In this study 4 new, highly purified porcine insulin preparations (ORGANON) were characterized by their time action profiles and in vitro and in vivo miscibility. These insulin preparations were soluble insulin, and suspensions of amorphous zinc insulin, mixture of 30% amorphous and 70% crystalline zinc insulin and NPH insulin. The time action profiles, assessed with the euglycaemic clamp technique and measurements of plasma insulin levels, in healthy volunteers were very similar to corresponding, already available, insulins of other manufacturers. In vitro miscibility was assessed for the soluble insulin with the 3 intermediate acting insulins in mixing ratios varying from 1:1-1:5. The recovery percentages of added soluble insulin, 75 sec after mixing in a 1:1 ratio with NPH, Tardum and Sub Tardum insulin, were 97.1%, 67.8% and 42.4%, respectively. The recovery of added soluble insulin decreased significantly with time of contact and with lowering of the mixing ratio for all insulins tested. In vivo insulin miscibility was performed for soluble and NPH insulin in a mixing ratio of 2:3, administered immediately after mixing in the syringe. The insulin action profiles were not altered when soluble and NPH insulin were administered after mixing as compared with the separate injections into contralateral thighs. In conclusion, the pharmacokinetics of these highly purified porcine insulins are in agreement with corresponding already available insulins. NPH insulin can be mixed with soluble insulin without affecting the absorption kinetics of either insulin.

Evaluation of urinary biomarkers for radical-induced liver damage in rats treated with carbon tetrachloride.

Carbon tetrachloride (CCl4) is a model compound for inducing free radical damage in liver. In this study 10 biomarkers in rats treated i.p. with three different single doses of CCl4 (0.25, 0.50, and 1.00 ml/kg body wt) were measured dose and time dependently and compared to evaluate these urinary products as noninvasive biomarkers for radical damage. Eight degradation products of lipid peroxides, namely, formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal, and malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and coproporphyrin III were measured in this study. As general measures of toxicity, several clinical chemical parameters (n = 12) and histopathological damage were determined. A dose-dependent increase in both the clinical parameters and the lipid degradation products was found. Increases in lipid degradation products were statistically significant at doses of 0.5 and 1 ml/kg CCl4. An increase in these products was already found in the first 12 h after exposure. At the lowest dose, 0.25 ml/kg CCl4, acetaldehyde and propanal already showed a statistically significant increase as well. No change in the urinary levels of 8-OH-dG could be found in this study and a decrease in the urinary excretion of coproporphyrin III was found. It is concluded that 8-OH-dG and coproporphyrin III are not useful biomarkers for radical damage induced by CCl4. Lipid degradation products, however, are promising noninvasive biomarkers for in vivo radical damage, although the precise specificity of these biomarkers for damage induced by radicals needs to be further investigated.